Confocal microscope with positionable imaging head

ABSTRACT

A confocal microscope for imaging tissue having an imaging head for capturing optically formed microscopic sectional images of tissue samples, a platform upon which is disposed a linear stage for moving the imaging head along a vertical dimension, and a rotary stage to rotate the linear stage and imaging head about the vertical dimension. A mounting arm couples the imaging head to the linear stage to adjust tilt of the imaging head and to rotate the imaging head about a normal axis perpendicular to an optical axis of an objective lens of the imaging head. In a first mode of operation, the imaging head is positioned to image an ex-vivo or in-vivo tissue sample upon the platform, such as ex-vivo tissue sample mounted upon a movable specimen stage, and in a second mode of operation the imaging head is positioned to image an in-vivo tissue sample beside the platform.

This application is a continuation of U.S. patent application Ser. No.15/810,093, filed Nov. 12, 2017, now U.S. Pat. No. 10,935,778, whichclaims priority to U.S. Provisional Patent Application No. 62/421,270,filed Nov. 12, 2016, which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a confocal microscope having apositionable imaging head mounted to a platform, and particularly to, aconfocal microscope having an imaging head mounted to a platformpositionable in one mode to image tissue samples disposed upon theplatform, such as an ex-vivo tissue specimen on a movable specimenstage, and in another mode to image tissue samples beside the platform,such as in-vivo skin tissue of large animals or humans.

BACKGROUND OF THE INVENTION

Confocal microscopes optically section tissue to produce microscopicimages of tissue sections without requiring histological preparation ofthe tissue on slides (i.e., slicing, slide mounting, and staining). Suchsectional images produced may be on or under the surface of the tissue.An example of a confocal microscope is the VivaScope® manufactured byCaliber Imaging & Diagnostics, Inc. of Henrietta, N.Y. Examples ofconfocal microscopes are described in U.S. Pat. Nos. 5,788,639,5,880,880, 7,394,592, and 9,055,867. In particular, U.S. Pat. No.7,394,592 describes an imaging head of a confocal microscope mounted ona multi-positionable arm extending from an upright station having acomputer system connected to the imaging head, where the computer systemshows on a display confocal images captured by the microscope. Whileuseful for imaging in-vivo tissue, such as a skin lesion without removalfrom a patient, it is cumbersome when one wishes to image ex-vivo tissuesamples as may be mounted on a microscope stage. Other confocalmicroscopes have been developed for use in imaging ex-vivo tissuesamples, such as may be mounted in tissue cassette holders, as describedin U.S. Pat. Nos. 6,411,434, 6,330,106, 7,227,630, and 9,229,210. Itwould be desirable to provide a confocal microscope from a commonplatform which can be used both for imaging ex-vivo tissue samplesmountable upon a stage and in-vivo tissue samples of a patient oranimal.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide aconfocal microscope having an imaging head supported from a platformwhere the imaging head is positionable over the platform or beside theplatform as desired for imaging tissue samples, thereby enabling ex-vivoimaging of tissue samples as may be disposed on a stage upon theplatform and in-vivo imaging of tissue samples as may be disposed besidethe platform.

Briefly described, the present invention embodies a microscope forimaging tissue having an imaging head with an optical system forcapturing optically formed microscopic sectional images, and a platformupon which is disposed first and second stages. The first stage iscoupled to the imaging head for moving the imaging head along a verticaldimension perpendicular to a horizontal dimension along which theplatform extends, such as to adjust a height of the imaging head. Thesecond stage rotates the imaging head about the vertical dimension. Theimaging head is positionable using at least the first and second stagesin a first mode to image at least a first tissue sample disposed betweenthe imaging head and the platform (i.e., upon the platform), and in thesecond mode to image at least a second tissue sample disposed beside theplatform (i.e., not upon the platform). Preferably, the optical systemis operative by confocal microscopy, such that the microscope of thepresent invention is referred to as a confocal microscope, but othermodalities for capturing optically formed microscopic sectional imagesmay be used.

In the first mode of the microscope operation, the first tissue samplemay be an ex-vivo tissue specimen (e.g., excised from a patient/subject)or in-vivo tissue of small animal or subject disposed upon the platform.While in the second mode of microscope operation, the second tissuesample may be in-vivo skin tissue of a human or large animal subject.

The optical system of the imaging head comprises optics having at leastan objective lens for focusing and collecting illumination from thefirst and second tissue samples when each face the objective lens. Thesecond stage is preferably a rotary stage between the first stage andthe platform for rotating the first stage and the imaging head coupledthereto 360 degrees about the vertical dimension. The first stage may bea linear slide stage having a carriage movable along the verticaldimension, in which such carriage is coupled to the imaging head by amounting arm. The mounting arm has a first portion fixed to thecarriage, and a second portion having a rotary stage for rotating theimaging head about a normal axis perpendicular to the optical axis ofthe objective lens. The second portion is further tiltable with respectto the first portion to adjust tilt of the imaging head along the normalaxis with respect to the horizontal dimension. The rotary stage and tiltadjustment provided by the mounting arm, and the first and secondstages, provides the imaging head with multiple (four) degrees offreedom of motion so it can be set by a user to different positions toimage ex-vivo or in-vivo tissue samples from a common platform uponwhich the imaging head is mounted in either first or second modes.

A third stage, such as an x-y stage, may be mounted to the platformmovable along x and y orthogonal axes along the horizontal dimensionalong which the platform extends. The first tissue sample, such asex-vivo tissue, is mounted upon such third stage for moving the tissuesample with respect to the objective lens during first mode operation ofthe microscope. The optical axis of the objective lens is aligned toextend along a z axis perpendicular to the x and y axes. This may beachieved by one or more of the above described rotation about the normalaxis and tilt of the imaging head until the optical axis aligns alongthe z axis. An optional fourth stage may be mounted to the third stagemoveable along such z axis, where the first tissue sample is mountedinstead upon the fourth stage to enable the tissue sample to be movableusing the third and fourth stages along x, y, and z axes with respect tothe objective lens. While the objective lens is movable within theimaging head along its optical axis, the fourth stage can provideadditional control for positioning the tissue sample along the z axis.The microscope may further be used in the first mode with the third andfourth stages removed from the platform, if desired. The third stage(and fourth stage if mounted thereto) may be referred to herein as aspecimen stage.

The microscope has a computer system connected to the imaging head toreceive signals representative of the images of the first or secondtissue samples when imaged. The computer system shows on a displayand/or store in its memory the images captured by the microscope. Thecomputer system controls operation of the imaging head responsive to auser via user interface device(s) provided. Movement of x and y axismotors of the third stage, and z axis motor of the fourth stage (ifpresent), are also preferably controlled by the computer system, but mayalternatively be controlled by a joystick if provided.

The optical system in the imaging head can utilize multiple discretelaser wavelengths for illumination, and selectable wavelengths fordetection. However, a single wavelength of laser illumination anddetection may be used. The objective lens may be removably mounted tothe microscope, such as by magnetics, so that different objective lensmay be mounted thereto, as desired by a user.

In the preferred embodiment, the objective lens of the optical systemfocuses and collects scanned illumination from a tissue sample, wherethe scanned illumination travels along a first path via the objectivelens to the tissue sample, and collected return illumination travelsalong a second path via the objective lens. The second path has at leasta beam splitter that splits the return illumination into first andsecond beams. The first beam travels to a first detector via a firstpinhole and a first selected position of an optical filter or opening(such along of a first filter wheel), and the second beam travels to asecond detector via a second pinhole and a second selected position ofanother optical filter or an opening (such along of a second filterwheel). One or both of the first pinhole and second pinhole are eachseparately adjustable in position to align their first and second beams,respectively, onto their first detector and second detector,respectively.

The optical system may further have a mirror in the second path toreflect the return illumination onto the beam splitter. Such mirror maybe adjustable in position to align the first beam when split by the beamsplitter onto the first detector via the first pinhole, which may thenbe non-adjustable in position, and the second pinhole is adjustable inposition to align the second beam via the second pinhole onto the seconddetector. Alternatively, the mirror may be non-adjustable in position,and one (or preferably) both the first and second pinholes are eachseparately adjusted in position to align their respective first andsecond beams onto their respective first and second detectors.

As the illumination of the tissue sample is of multiple discretewavelengths, the first and second detectors receive differentwavelengths of the collected illumination to enable simultaneous captureof a same one of the images at the different wavelengths or wavelengthrange on the first and second detectors in accordance with the firstselected position and the second selected position having at least oneof the optical filter and the another optical filter, respectively.Where one or more of the discrete wavelengths of illumination canactivate fluorescent dye(s) that may be applied to tissue sample, theoptical filter in the path of one of the first or second beams isselected to enable detection of fluorescent wavelength(s) associatedwith the dye(s) on their associated detector. Where non-fluorescentimaging is desired, an open position is selected in the path of one ofthe first or second beams to detect light of a discrete wavelength ofillumination that was present along the first path to the tissue sample.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing objects, features, and advantages of the invention willbecome more apparent from a reading of the following description inconnection with the accompanying drawings, in which:

FIGS. 1 and 2 are two perspective views taken from different angles ofthe imaging head of the microscope of the present invention mounted to aplatform, where FIG. 1 shows an example of an ex-vivo tissue samplebeing imaged and FIG. 2 shows an example of an in-vivo tissue samplebeing imaged;

FIG. 3 is another view of the imaging head of FIG. 1 and the platform inwhich the assembly of a rotary stage and a vertical stage are shownexploded from the platform, and an x-y stage is shown with an explodedoptional z-stage mountable upon the x-y stage;

FIG. 4 is another view of FIG. 1 with the z axis stage of FIG. 3 shownmounted upon the x-y stage, and the imaging head removed;

FIG. 5 is a partial cross-sectional view of FIG. 1 taken along line 5-5in the direction of arrows at the end of the line showing the mountingarm which couples the imaging head to a carriage of the vertical stage;

FIG. 6 is another view of FIG. 1 in which the assembly of the mountingarm of FIG. 5 is shown exploded between the imaging head and thecarriage of the vertical stage, where the z-stage of FIG. 3 is mountedto the x-y stage;

FIG. 7 is another exploded view of the assembly of the mounting arm ofFIG. 5 , but from a different angle of that of FIG. 6 , and having therotary stage, vertical stage, and platform removed;

FIG. 8 is a block diagram of the microscope of the present inventionhaving an imaging head supported over a platform as shown in FIG. 1 witha computer system, a display for showing images captured by themicroscope, and a multi-wavelength laser light source which may provideadditional imaging wavelengths of light, as well as other components forcontrolling position of the vertical stage, x-y stage, and z-stage;

FIG. 9 is an exploded perspective view of the imaging head of FIG. 1apart from the rest of the microscope of FIG. 8 ;

FIG. 10 shows the imaging head of FIG. 9 assembled with the housing ofthe imaging head removed; and

FIG. 11 is an optical diagram of the optical system in the imaging headof FIG. 1 .

DETAILED DESCRIPTION ON THE INVENTION

Referring to FIGS. 1, 2, and 3 , an imaging head 12 of a microscope 10(FIG. 8 ) is shown in a housing 13 supported over a platform (or base)14 having an upper surface 14 a along a horizontal or dimension orplane. A second (or rotary) stage 16 is mounted to platform 14 forrotating a first (or vertical) stage 18 about a vertical dimension,i.e., perpendicular to the horizontal dimension along which uppersurface 14 a of platform 14 extends. Vertical stage 18 is a verticallydisposed linear slide stage which carries a movable carriage 20 fortranslation along such vertical dimension. Carriage 20 is coupled by amounting arm 22 to a base 15 of housing 13 of imaging head 12. Themounting arm 22 enables adjustment of tilt and rotation of the imaginghead 12 at a desired adjustable rotational position and height positionas set by rotary stage 16 and vertical stage 18, respectively, as willbe described later below in more detail.

The imaging head 12 has an optical system 11 for capturing opticallyformed microscopic sectional images of tissue samples. The operation andstructure of imaging head 12 may be the same as the confocal microscopeof U.S. Pat. No. 9,055,867 which is incorporated herein by reference,but the preferred optical system 11 is shown in FIGS. 9-11 . The opticalsystem 11 has an objective lens 128 within an extending snout 127 of theimaging head 12 for focusing and collecting illumination from tissuesamples facing the objective lens. Objective lens 128 has an opticalaxis 128 a, and perpendicular to such optical axis 128 a is a virtualnormal axis 128 b. Axes 128 a and 128 b are depicted as dashed lines inFIGS. 1 and 2 .

Rotary stage 16 has a base 24, which is mounted to platform 14 by screws25 via threaded holes 26 (FIG. 3 ) in platform 14, and a turntable 28which rotates with respect to base 24, as indicated by arrow 30,responsive to rotation of a graduated knob (or hand crank) 29 to gearing(not shown) disposed to rotate turntable 28. A locking pin 27 may slideor be turned to move in and out with respect to a hole in base 24 inorder to lock and unlock, respectively, the rotational position ofturntable 28 with respect to base 24. Turntable 28 is rotatable 360degrees and may have graduations along its outer circumference inrotational degrees with respect to marking(s) along base 24, which maybe utilized by a user when manually turning knob 29 clockwise orcounterclockwise to effect desired rotation. The rotary stage 16 ispreferably a Velmex rotary table, model no. A4872TS (manufacturer:Velmex, Inc., Bloomfield, N.Y. USA), but other rotary tables may also beused. A circular adapter plate 32 is mounted atop turntable 28 by screws33 received in threaded holes 34 of the adapter plate 32 and threadedholes 35 of turntable 28. Adapter plate 32 may be made of stainlesssteel.

Vertical stage 18 has a housing 36 that extends upwards from rotarystage 16 and platform 14. Carriage 20 is received along an open side 36a of housing 36 and mounted for vertical translation between sides 36 band 36 c of housing 36. As best shown in FIG. 4 , housing 36 has abottom wall 40 and a top wall 41 between which is journaled are two endsof a rotatable vertical lead screw 42 that extends through a verticalopening 46 of carriage 20. Carriage 20 has two vertical slots or grooves43 that ride along two inwardly protruding rails 44 from opposing sides36 b and 36 c of housing 36. Lead screw 42 extends along a threaded hole49 of a nut 48 (shown in dashed lines) fixed in carriage 20. The threadsof lead screw 42 engage the threads of nut 48 along hole 49 so thatrotation of lead screws 42 moves nut 48 and thus carriage 20 attachedthereto up and down vertically along rails 44. Rotation of the leadscrew 42 in a first direction causes the carriage 20 to move upwards,and in a second direction causes the carriage to move downwards, asindicated by arrow 45. A stepper motor 50 is mounted along the top ofhousing 36 and extends through an opening in top wall 41 to engage thetop end of lead screw 42. Motor 50 control the rotation and direction ofrotation of lead screw 42 and thus the vertical height of carriage 20with respect to the horizontal dimension along which platform 14extends. Vertical stage 18 is preferably a Velmex BiSlide® model numberMN-0100-M02-21, but other vertical stages may also be used. Steppermotor 50 may be a Vexta Stepper Motor Model No. PK266-03A-P1(manufacturer: Oriental Motor Co. Ltd., Japan), but other stepper motorsmay be used. To mount vertical stage 18 to turntable 28 of rotary stage16, bottom wall 40 of housing 36 has holes 54 (FIGS. 3 and 4 ) throughwhich four screws 55 extend into threaded holes 56 of the adapter plate32, where two screws 55 are shown in FIG. 3 , and the other two screws55 are shown in FIG. 4 .

Two limit switches 52 are provided each having a switch element 52 awhich actuates when abutted by an extension 53 from carriage 20 todefine the uppermost extent and lowermost extent of carriage 20 travelup and down, respectively. When actuated, the limit switch 52 sends asignal to a below discussed controller 134 to turn off motor 50operation to avoid over travel of carriage 20 in housing 36.

The mounting arm (or assembly) 22 coupling carriage 20 to housing 13 ofimaging head 12 will now be described and is best shown by thecross-sectional view of FIG. 5 , and exploded views of FIGS. 6 and 7 .An adapter mount plate 58 is attached to a slanted/angled support memberor plate 60 by a screw 61 via hole 62 in plate 58 and threaded hole 63in support member 60, where pins 64 extend from support member 60 andalign with holes 65 of adapter mount plate 58. A tray member 68 isattached to support member 60 by a screw 70 via a hole 69 of tray member68 received in a threaded hole 71 of support member 60, where pins 72extend from support member 60 and align with holes 74 in the bottom oftray member 68.

Tray member 68 receives a tilt plate 76 which has been attached byscrews 77 via holes 78 in tilt plate 76 into threaded holes 79 of a base80 a of a rotary stage 80, which has a turntable 80 b rotatable withrespect to base 80 a. Two ball bearings 66 are disposed in tray member68 between the inside of tray member 68 and bottom of tilt plate 76.Such ball bearings 66 are each glued in a semicircular pocket along theinterior bottom of tray member 68 prior to receiving tilt plate 76 withattached rotary stage 80 in order to provide two points of contact withtilt plate 76 near the top left and top right of tray member 68. Fourscrews 82 extend through holes 83 in tilt plate 76 and holes 84 in traymember 68 and are each captured by one of four nuts 86 via one of foursprings 85. Springs 85 and nuts 86 are also shown in FIGS. 1-3 . Springs85 bias tilt plate 76 toward the inside bottom of tray member 68 withtwo ball bearings 66 being disposed there between. An adapter mountplate 88 is attached by screws 89 via holes 90 in plate 88 to threadedholes 91 along turntable 80 b of rotary stage 80. Screws 92 extendthrough holes 94 in plate 88 into threaded holes 95 (FIG. 7 ) along base15 of housing 13 of imaging head 12. The mounting arm 22 is mounted byfour screws 96 which extend via holes 98 of plate 58 into threaded holes100 (FIG. 6 ) for receiving such screws 96 along carriage 20. Theslant/angled support plate 60 is set at a desired upward angle or slopeto dispose the rest of the mounting arm 22 mounted to plate 60 at ahigher height than would be if support plate 60 was horizontallydisposed in order to obtain a desired range of vertical travel ofimaging head 12 as set by the position of switches 52 along housing 36.However, other angle or non-angled (i.e., horizontal) support member maybe used depending on the desired height of vertical stage 18, and therange of vertical travel of carriage 20 as set by limit switches 52. Forpurposes of illustration, only one of each set of screws, springs,holes, and nuts, are labeled in FIGS. 6 and 7 .

As best shown in FIG. 5 , a thumb screw 102 with attached handle or knob102 a is provided to adjust tilt of plate 76 with respect to tray member68. Thumb screw 102 extends through a threaded hole 103 near the centerbottom of plate 76 against an opening 104 along the inside of traymember 68 which abuts the end of thumb screw 102. The thumb screw 102rotation changes the distance of the bottom of plate 76 with respect tothe bottom of tray member 68 under the bias of springs 85, while theball bearings 66 provide two points of contact upon which plate 76tilts, thereby controlling the tilt or yaw (or tilt angle) of the normalaxis 128 b (FIGS. 1 and 2 ) of the housing 13 of imaging head 12 withrespect to the horizontal via the attached rotary stage 80, as denotedby arrows 105 (FIG. 1 ). The tilt position of the imaging head 12 maythus be varied from the horizontal as desired by the user, such as forexample at or between 0 to 5 degrees. Tray member 68, plate 76, adapterplates 58 and 88, and support member 60 may be made of aluminum.

Rotary stage 80 operates to rotate turntable 80 b with respect to base80 a responsive to rotation of a knob or micrometer 81 to gearing (notshown) disposed to rotate turntable 80 b as indicated by arrow 106 (FIG.1 ) about the normal axis 128 b. A locking pin 107 may slide or beturned to move in and out with respect to a hole in base 80 a in orderto lock and unlock, respectively, the rotational position of turntable80 b with respect to base 80 a. Turntable 80 b is rotatable 360 degreesto rotate imaging head 12 about normal axis 128 b and may havegraduations along its outer circumference in rotational degrees withrespect to marking(s) along base 80 a, which may be utilized by a userwhen manually turning knob 81 clockwise or counterclockwise to effectdesired rotation. The rotary stage 80 is preferably a Newport PrecisionRotation Stage Model No. UTR80 (manufacturer: Newport Corporation,Irvine, Calif., USA), but other rotary stages may also be used. Thus,plate 58, support member 60, and tray member 68 extend along a firstportion of mounting arm 22 which is fixed to carriage 20 as describedabove, and tilt plate 76, rotary stage 80, and plate 88 extend along asecond portion of mounting arm 22 coupled to the imaging head 12, asdescribed above, so that the second portion tilts with respect to thefirst portion by tilting plate 76 in tray member 68 of the first portionto adjust tilt of the imaging head 12 along normal axis 128 b withrespect to the horizontal dimension along which surface 14 a of platform14 extends.

In summary, the entire mounting arm 22 with imaging head 12 can rotatewith vertical stage 18 using rotary stage 16 (arrow 30) to a desiredrotational position about the vertical dimension (with locking pin 27temporarily released until new rotational position is reached) usinghand crank 29. The entire mounting arm 22 with imaging head 12 can beset to a desire height or distance along the vertical dimension usingvertical stage 18 (arrow 45) from the horizontal dimension along whichsurface 14 a of platform 14 extends. Thumbscrew 102 of the mounting arm22 can be turned using handle 102 a to adjust tilt plate 76 to a desiredtilt position with respect to tray member 68 (arrow 105), and imaginghead 12 can rotate using rotatory stage 80 (arrow 106) to a desiredrotational position about normal axis 128 b that extend through of theimaging head 12 and along the rotational axis of rotary stage 80 aboutwhich turn table 80 b rotates (with locking pin 107 temporarily releaseduntil new rotational position is reached). This freedom of motion alongarrows 30, 45, 105, and 106 allows imaging head 12 in a first mode ofoperation of microscope 10 to be moved to a position, such as shown inFIG. 1 to image, for example, a first (or ex-vivo) tissue sample 110between imaging head 12 and platform 14, and in a second mode ofoperation of microscope 10 moved to a position such as shown in FIG. 2to image, for example, a second (or in-vivo) tissue sample 113 of apatient/subject beside (i.e., at the side of, nearby, but not upon)platform 14. In-vivo tissue sample 113 has, for example, a skin lesion113 a. The patient/subject is not shown to scale in FIG. 2 .

As stated earlier, objective lens 128 extends in snout 127 from housing13. Snout 127 may have an optional snout cover 127 a with a materialplate window 127 b of optically transparent material, such as glass orplastic. Preferably, window 127 b is thick, such as 1 mm. Cover 127 a isshaped to extend over snout 127 and objective lens 128 with imagingbeing carried out through window 127 b. It is especially useful so thatpressure may be applied by the window 127 b against a surface of tissuebeing imaged, such as an in-vivo tissue sample, to assist instabilization of the optical system 11 of the imaging head 12 to suchtissue to improve imaging performance. The snout cover 127 a and window127 b may also be used to image in-vivo samples, such as small animals,that may be placed upon platform 14 with or without specimen stage(s)present.

In such first mode of operation, ex-vivo tissue sample 110 may bemounted onto a movable specimen stage provided by a third (or x-y) stage108 movable along x and y orthogonal axes (depicted as x and y arrows inFIG. 1 ) which are parallel to the horizontal plane of surface 14 a ofplatform 14 upon which stage 108 is mounted. For example, ex-vivo tissuesample 110 may a non-histologically prepared tissue specimen (i.e.,without being mechanically sliced thin sections mounted on slides)removed from a patient/subject which is disposed upon a block 111. Block111 may represent a substrate, such as of glass or plastic, or acassette which retains the tissue sample 110 in a desired orientation onstage 108. Mounting features or inserts 109 receive block 111, and clips112 retain block 111 position in stage 108. However, other mechanismsfor retaining block 111 may be used. While stage 108 may be a typicaltranslation stage for moving tissue sample 110 along x and y orthogonaldimensions, preferably stage 108 is a Marzhauser X-Y Stage Scan^(Plus)Model No. 00-24-579-0000 (manufacturer: Märzhäuser Wetzlar GmbH & Co.KG, Germany). Stage 108 is mounted to platform 14 as typically inmounting stages to bases, such as by screws 109 a through holes in stage108 in stage mounts 108 a attached to platform 14. Preferably, two stagemounts 108 a are used as shown in FIG. 2 between stage 108 and platform14. Stage mounts 108 a may be attached to platform 14 by screws viaholes through such mounts 108 a and platform 14. Rubber o-rings andwashers (such as tooth lock washers) may be disposed along such screws109 a in attaching the stage 108 and stage mounts 108 a (FIG. 2 ), inwhich o-rings aid in minimization of vibration to block 111 holdingtissue sample 110. Other mechanisms for coupling stage 108 to platform14 may also be used. Four rubber feet 115 are attached to the undersideof platform 14, such as by screws through holes in the platform, so thatthe platform may rest on a tabletop or other surface upon such rubberfeet.

Optionally, an additional fourth (or z) stage 114, such as shown inFIGS. 3, 4, and 6 , may be attached atop stage 108 in which the mountfor tissue sample 110 as shown in FIG. 1 is removed and placed uponstage 114 instead. Stage 114 is movable along a z axis orthogonal to thex and y axes of stage 108. Stage 114 preferably is a Marzhauser PiezoZ-Stage Model No. 00-55-550-0800, which attaches by clips onto stage108, for coupling such stages as set forth by the manufacturer.

Referring to FIG. 8 , an example of microscope 10 in a desktop or tableconfiguration is shown having platform 14 disposed upon a surface 116with imaging head 12 mounted to platform 14 as described above to enableboth first and second modes operation of the microscope. The microscope10 has a computer system 118, such as a personal computer or workstationprogrammed in accordance with software in its memory. Computer system118 is connected to a display 120 and user interface devices (such askeyboard 121 and mouse 122). Display 120 may be a touchscreen displaywhich provides an additional user interface device to graphical userinterface software operating on computer system 118. Computer system 118is connected by a cable 124 to imaging head 12, and via such cable thecomputer system controls operation of imaging head 12 and receivesignals therefrom representative of one or more microscopic images ofoptically formed tissue sections at one or more locations within or upontissue sample 110 or 113 for output to display 120 and storage in memoryof the computer system in the same manner as VivaScope® confocalmicroscopes manufactured by Caliber Imaging & Diagnostics, Inc. ofHenrietta, N.Y., USA. The location of the platform 14 upon surface 116may be different than shown in FIG. 8 so that imaging head 12 can beproperly positioned with respect to a patient or subject that needs tolocated beside both surface 116 and platform 14 in order to imagein-vivo tissue of such patient or subject in the second mode operationof microscope 10.

In microscope 10 operation, scanned laser illumination is focused andcollected by objective lens 128 along its optical axis 128 a into tissuesample 110 or 113, where collected light by the lens is representativeof a tissue section at a cellular level below the surface of the tissuesample facing objective lens 128. While FIG. 8 shows an example of firstmode operation for imaging an ex-vivo tissue sample of FIG. 1 , thehousing 13 is movable for imaging an in-vivo tissue sample as describedearlier for second mode operation of microscope 10 as shown in FIG. 2 .As stated earlier, tissue samples may also be positioned on platform 14for imaging by imaging head 12 without stage(s) 108 or 114 present, suchas may be useful for imaging in-vivo tissue samples of small animals.

The electronics of imaging head 12, and the computer system 118 ofmicroscope 10 with display 120, for viewing microscopic sectional imagesof tissue samples from light focused and collected via objective lens128, may be the same as described in incorporated by reference U.S. Pat.No. 9,055,867. While in FIG. 8 both stages 108 and 114 are shown whenimaging ex-vivo tissue samples, z-stage 114 is optional, since objectivelens 128 is movable in housing 13 along its optical axis 128 a which canbe aligned with a z axis orthogonal with the x and y axes of stage 108so that objective lens 128 is thus movable along such z axis in order toselect the depth of focus of a beam scanned at locations upon or withinthe tissue sample being imaged. However, when z-stage 114 is presentupon platform 14, z-stage 114 may also be used to select the depth offocus a beam scanned at locations upon or within the tissue sample beingimaged. Imaging head 12 is temporarily fixed in position with respectwith respect to the x and y axes of x-y stage 108 in the first mode ofoperating microscope 10.

Computer system 118 controls movement of x, y motors of the stage 108and reading x and y positions thereof, and z axis motor and reading zportion thereof of stage 114 (if present), via one or more cables 130 toports 129. FIG. 3 shows ports 129 with cable 130 removed. Preferably astage controller card is provided in the computer system 118 to enablesuch interface with stages 108 (and 114 if present) via cable(s) 130. Inthe case of Marzhauser X-Y Stage and Marzhauser Z-Stage as describedearlier are utilized, such stage control card may be a Scan^(Plus)Marzhauser X&Y Stage Controller Card, Part Number 00-76-150-0813,located inside the case of computer system 118. An optional joystick132, may also be used by a user to control movement of x, y axis motorsof the stage 108 (and z axis motor of stage 114 if present), via a cable133 to one of ports 129.

The stepper motor 50 of vertical stage 18 is controlled by a motorcontroller 134 which drives motor 50 via signals along a cable 136 torotate lead screw 42 of the vertical stage 18 in first and seconddirections that enable up and down motion, respectively, of carriage 20and the imaging head 12 mounted thereto by mounting arm 22. Limitswitches 52 are also connected to motor controller 134 via cable 136 toreceive signals therefrom and control motor 50 accordingly as describedearlier. Buttons and switches 135 along the motor controller 134 areprovided to control motor operation. Preferably, motor controller 134 isa Velmex Stage Controller Model No. VXM-1. An optional joystick 138 mayconnected by cable 139 to motor controller 134 to facilitate usercontrol of motor 50 to provide desired up and down motion of carriage 20of vertical stage 18. Preferably, joystick 138 is a Velmex DigitalJoystick Model No. 4-2121. While rotary stage 16 is shown as beingmanually controlled by knob 29, optionally stage 16 may have a motorinstead of knob 29 for rotating rotary stage 16, such as manufactured byVelmex. This optional motor may be controlled by signals from motorcontroller 134 which can additional drive such motor. Although notshown, power is supplied to various components in FIG. 8 to enable theiroperation.

In operating microscope 10 using stage 108, the optical axis 128 a ofobjective lens 128 is aligned along a z axis perpendicular to the x andy axes of stage 108, such as shown in FIG. 1 . This further enablesalignment along the z axis of stage 114 if present. Such alignment isenabled by adjusting the tilt of imaging head 12 using handle 102 a ofthumb screw 102 and rotation of imaging head 12 using rotary table 80,as described earlier. Such may be aided by alignment mark(s) if presentalong rotary table 80 along turntable 80 b and base 80 a. Further, atarget or features may be placed on the platform 14, or stage mount (inplace of block 111 of FIG. 1 ), in view of objective lens 128 to assistin electronic calibration of images on screen 125 of display 120 as suchcalibration alignment is carried out to assure horizontal levelling ofimaging head's normal axis 128 b with respect to platform 14, where suchnormal axis 128 b lies perpendicular to the z axis.

While the operation and structure of imaging head 12 may be the same asdescribed in incorporated U.S. Pat. No. 9,055,867 using the laserillumination source provided therein (such as light source 146 of FIG. 9), the imaging head of the incorporated patent preferably is adapted tothat of optical system 11 (FIGS. 9-11 ) which utilizes multiple discretelaser wavelengths for illumination provided from a multiple wavelengthlaser light source 140 via a fiber optic cable 142 to imaging head 12.For example, light source 140 may be a Toptica iChrome MLE-L Multi-LaserEngine (manufacturer: Toptica Photonics AG, Germany) with collimatedlaser diode assemblies manufactured by Blue Sky Research of Milpitas,Calif. USA. The additional laser illumination provided by light source140 is combined with the laser illumination produced in imaging head 12and scanned together via objective lens 128, and then returned scannedillumination via objective lens 128 is split for detection onto twodetectors that sense particular wavelength(s), as described in moredetail below in connection with FIGS. 9, 10 , and 11.

Referring to the optical system 11 of FIG. 11 , linear polarized lightof multiple discrete wavelengths generated by light source 140 (e.g.,405 nm, 488 nm, 561 nm, and 640 nm) passes along optical fiber cable 142to optics 143 which collimates and expands its size to provide a beam144, such as to 4.3 mm in diameter. Optics 143 are preferably containedin a cylindrical tube 143 a that receives optical fiber cable 142 andextends through an opening 13 a (FIG. 1 ) of housing 13. A laser 146,preferably a laser diode which is associated with an opto-detector formonitoring laser power as described in the incorporated patent, providesa linearly polarized beam 148 at a single wavelength (e.g., 785 nm).Beam 144 and beam 148 are combined into a beam 150 by a dichroicbeamsplitter 149, and beam 150 then passes through a polarizingbeamsplitter 151. A resonant scanner 152 presents its scanning mirror152 a to beam 150, and the beam from the resonant scanner mirror 152 ais then incident scanning mirror 154 a of a galvanometer 154 to providea scan beam 155. Mirrors 152 a and 154 a oscillate so that mirror 152 aprovides fast or horizontal line scans in a raster being scanned, andslow or vertical scan and retrace are provided by mirror 154 a, asdescribed in more detail in the incorporated patent. The axes ofoscillation of these mirrors 152 a and 154 a are orthogonal(perpendicular) to each other. The separation distance may beapproximately a minimum separation distance to provide clearance betweenthe mirrors 152 a and 154 a as they scan. A telescope 156 magnifies thebeam (e.g., 2.3 x) and relays scanning beam 155 to objective lens 128via a quarter wave plate shifter 157, and the objective lens 128 focusesthe scanning beam 155 to the sample, such as sample 110 or 113 forexample as earlier described.

The returned light 158 from the tissue sample 110 or 113 passes throughobjective 128, wave plate 157, telescope 156, and scanning mirrors 154 aand 152 a. The return light thus is descanned at mirrors 154 a and 152 ainto a stationary beam 160 and enters the polarizing beamsplitter 151which reflects beam 160 via a focusing lens 161, a reflecting mirror162, and a notch filter 163, to a dichroic beamsplitter 164 which splitsthe returned light into a first beam 165 and a second beam 169. Beam 165is incident a small aperture provided by pinhole 166 onto a detector 168provided by a photomultiplier tube, via one of selectable open or filterpositions along a filter wheel 167. Beam 165 is incident a smallaperture provided by a pinhole 170 onto a detector 172 provided by aphotomultiplier tube, via one of selectable open or filter positionsalong a filter wheel 171. Lens 161 focuses the light of their respectivebeams onto pinholes 166 and 170. Although not shown in FIG. 11 , aturning mirror 173 (FIG. 9 ) is provided between beamsplitter 151 andmirror 152 a to reflect beam 150 onto mirror 152 a, and reflect beam 160from mirror 152 a to beamsplitter 151.

Each of filter wheels 167 and 171 has a shaft mounted for rotation bystepper motors 174 and 175, respectively, to select the desire openingor filter along the wheel. For example, at least four filters areprovided along wheel 167 for different wavelength(s) or range ofwavelengths onto detector 168, where filter 167 a passes light only inrange of 405-561 nm wavelength, filter 167 b passes only 630 nmwavelength light, filter 167 c passes only 670 nm wavelength light, andfilter 167 d passes both 832 nm and 837 nm wavelength light. At leasttwo filters are provided along wheel 171, where a filter 171 a passesonly 520 nm wavelength light, and a filter 171 b passes only 450 nmwavelength light. Spaces on one or both filter wheels are open so thatunfiltered light may pass there through, such as for detection of lightof the wavelength of laser 146 in the path of light for detection bytheir respective detectors 168 and 172. For example, opening 167 e isprovided on filter wheel 167, and opening 171 c is provided on filterwheel 171. Additional openings/filters illustrated on filter wheel 171may have filters for other wavelengths or wavelength ranges.

The wavelengths provided along fiber optic cable 142 can activatefluorescent dyes that may be applied to tissue samples. Thus, one of thefilter wheels enables selection of a filter to detect on theirassociated detector the fluorescent wavelength(s) of the returned light158, while the other of the filter wheels is set to an open position todetect light of wavelength of laser 146 in the returned light 158. Notchfilter 163 allows selectable discrete wavelengths or ranges ofwavelengths to assist in detecting wavelengths with filters along thefilter wheels. Preferably, notch filter 163 allows light of wavelengthof laser 146 (e.g., 785 nm), and blocks light of wavelengths receivedfrom fiber optic cable 142 which may interfere with imaging atfluorescent wavelengths associated with the filters disposed alongfilter wheels 167 and 171 in the path of light for detection by theirrespective detectors 168 and 172. Further, dichroic beamsplitter 164 mayfilter light such that beam 165 has wavelengths 405 nm and 408 nm, and abeam 169 has wavelengths 581 nm, 640 nm, and 785 nm. Other wavelengthsthan set forth above may be used for light sources 140 and 146, anddetected beams 165 and 169, as well as other wavelength filtering may beused by notch filter 163 and along filter wheels 167 and 171.

Each motor 174 and 175 is driven by electronics on a printed circuitboard 185 having a Hall effect sensor which reads a magnet along thewheel to sense the home position of the wheel and rotate the wheel tothe desired filter or open location along the wheel by actuation signalsreceived from computer system 118. The rotational position of eachfilter or opening along filter wheels 167 and 171 may be stored memoryof the computer system 118 so that motors 174 and 175 can be actuated bycomputer system to arrive at the rotational position associated with thedesired filter or opening along the wheels.

The optical components and electronics of the imaging head 12 aremounted along a chassis 176 and support plate 177 as shown in FIGS. 9and 10 to provide a preferred compact mounting of such components. Twoprinted circuit boards 190 with electronics for controlling imaging head12, responsive to computer system 118, are attached to chassis 176,where circuit board 190 are connected to other circuit boards describedherein in housing 13. A first structure or block 178, such as ofaluminum, is mounted to chassis 176 which supports light source 146,beamsplitter 149, and cylinder 143 a with attached fiber optic cable142. Structure 178 has a receptacle 179 into which cylinder 143 a plugsinto when additional wavelengths for imaging from fiber optic cable 142is desired. Detectors 168 and 172 have a circuit board 182 and 183,respectively, which is mounted to support plate 177. A second structureor block 180, which may also be of aluminum, is mounted to chassis 176to support filter wheels 167 and 171, pinholes 166 and 170, beamsplitter164, and notch filter 163, for imaging onto such detectors 168 and 172as described earlier. The circuit board 185 for driving and controllingmotors 174 and 175 may be supported on circuit board 182.

In order to properly align beams 165 and 169 for detection, mirror 162and pinhole 166 are each adjustable in position. Mirror 162 is mountedupon an adjustable flexure 162 a attached to a bracket or flange 186 ofchassis 176 for steering beam 169 via beamsplitter 164. Flexure 162 amay be adjustable by screws, and for example may be a stainless steelflexure mirror mount, such as Flexure Industrial Optical Mount withAllen (or hex) key adjustments, model No. MFM-050 manufactured byNewport Corporation of Irvine, Calif., U.S.A. This adjustability ofmirror 162 spatial position is denoted by arrows beside mirror 162 inFIG. 11 . Pinhole 166 (i.e., provided by a thin substrate with lightblocking material having a small aperture) is retained in a cylinder (orcylindrical cell) 188 mounted in structure 180, where pinhole 166 isspring loaded using two spring steel flexures. Two orthogonally orientedset screws 189 push pinhole 166 into a desired position against thespring force of such flexures, so that turning screws 189 adjustspinhole 166 position. Optionally, such adjustability may be similarprovided in structure 180 in a third orthogonal dimension. Thisadjustability of pinhole 166 spatial position in two dimensionsorthogonal to the incident beam 165, or in three dimensions, is denotedby arrows beside pinhole 166 in FIG. 11 .

When imaging head 12 is assembled, mirror 162 is adjusted in position tosteer beam 169 in alignment with pinhole 170, which is fixed inposition, so that beam 169 detected by detector 172 can be properlyimaged. To aid in such alignment, an image is displayed on display 120by computer system 118 from detector 172, and adjustable mirror 162 ismoved until beam 169 is aligned with the fixed pinhole 170 such thathighest signal level from detector 172 is achieved on display 120. Thenan image from detector 168 is displayed on display 120, and adjustablepinhole 166 is moved until the highest signal from detector 168 isachieved on display 120. Thus, beam 165 is now aligned with pinhole 166so that beam 165 detected by detector 168 can be properly imaged.Alternatively, adjustable flexure 162 a is not used so that mirror 162is mounted non-adjustable in position when imaging head 12 is assembled,and pinhole 170 is manually adjustable in position in the same waypinhole 166 is disposed. In such case, one or preferably both pinholes166 and 170 are each separately adjusted in position to align theirrespective beams 165 and 169 onto their respective detectors 168 and 172by the highest signal being achieved on display 120 from theirrespective detectors 168 and 172.

The adjustability in alignment of beams 165 and 169 assures properoperation of dual detection path of optical system 11 of the beams ontodetectors 168 and 172, respectively, so that microscope 10 cansimultaneously provide images of microscopic structures of the sametissue sample using two different wavelengths or wavelength range ofdetected returned light from the sample. Filter wheels 167 and 171 areeach set to one of openings or filters accordingly, so that one or bothimages of the desired wavelengths or wavelength range can appear ondisplay 120 by computer system 118 from received signals of detectors168 and 172. Pinholes 166 and 170 may be identical, and they enableconfocal imaging on their respective detectors by limiting returnedscattered light of their respective beams to a particular section withinor on the tissue sample 110 or 113.

Attached to the forward end of chassis 176 is a fixed tube 192 withoptics providing telescope 156. Objective lens 128 is disposed in agenerally cylindrical mounting 194 that attaches to a barrel 196providing a tube or sleeve moving axially (along optical axis 128 a)over tube 192 by a linear actuator as described in the incorporatedpatent. A magnetic strip is provided on the side of barrel 196 which isread by a sensor 197 on chassis 176 that linearly encodes position ofthe barrel 196 to the electronics in the imaging head 12, therebyenabling computer system 118 to actuate the linear motor to adjust theposition of objective lens 128 with respect to tube 192 and hence thefocus of such lens with respect to the tissue sample 110 or 113.

Preferably, cylindrical magnets 198 are attached, such as by adhesive,to holes 199 along the interior annular ring 200 at end of barrel 196,as shown in FIG. 9 . A metal ring 201 is attached to the objective lensmounting 194. Such ring 201 attaches along ring 200 by magneticattraction to magnets 198, so that mounting 194 is retained to barrel196. The objective lens 128 shown in the figures represents a liquidimmersion lens. Such objective lens 128 is useful when a refractiveindex matching fluid is applied to a tissue sample prior to being imaged(the fluid matches or approximately matches the refractive index of thetissue sample) as the objective lens is brought into contact with thesurface of the tissue sample. The index matching fluid reducesundesirable reflections and spherical distortions from the tissuesample's surface facing the lens that can negatively effect imagingperformance. However, different objective lens may be provided indifferent mountings 194, each providing a different imaging performance,such as in terms of magnification, or are of non-immersion or differentimmersion type lenses. When a different objective lens 128 is desired bya user, the user can pull mounting 194 away for magnets 198, and replacewith a different mounting 194 with the desired objective lens.Alternatively, the mounting 194 may be attached such as by adhesive, tothe end of barrel 196, without metal ring 201 or magnets 198.

The earlier described snout 127 is provided by barrel 196 with attachedmounting 194 having objective lens 128. As shown in FIG. 9 , snout cover127 a is provided by a cylindrical tube having a window 127 b mounted ina cap 127 c that is received in an opening 127 e at the distal end ofsuch tube which is shaped to receive cap 127 c. To mount snout cover 127a to imaging head 12, a cylindrical mounting 202 is provided havingthree legs 203 that attach, such as by adhesive, to the front of chassis176 and extend via an opening 13 b at the front of housing 13 withbarrel 196. Along the front inner annular rim 204 of mounting 202 areholes 204 a for cylindrical magnets 205 which are retained in the holesby adhesive. A metal ring 127 d is attached at the rear of the tubeproviding snout cover 127 a. Attraction of ring 127 d to magnets 205along rim 204 retains snout cover 127 a in position for imaging throughwindow 127 b via objective lens 128. Snout cover 127 a may be removedfrom its mounting 202 by pulling cover 127 a away from magnets 205 whensnout cover 127 a is not needed for imaging.

Housing 13 has a series of ribs 208 extending from base 15 of housing 13onto which chassis 176 and plate 177 are mounted, such as by screws intoholes along such ribs. Left and right housing portions 206 and 207provide shells that mate with each other and attach to ribs 208 byscrews. Two handles 210 are then attached to housing portions 206 and207 to assist in manually moving housing 13 by a user if desired withrespect to platform 14. If a fan is provided in housing 13, a fan cover211 may be used. Other manner of coupling imaging head 12 componentswithin housing 13 may be used than shown in FIGS. 9 and 10 .

Computer system 118 via cable 124 has an I/O interface with electronicsin the imaging head 12 to enable their operation, such as to control ofoperation of resonant scanner 152 and galvanometer 154, control thelinear actuator or motor for positioning objective lens 128 along itsoptical axis 128 a, and power to light source 146, as may be describedin more detail in the incorporated patent. The signals from detectors168 and 172 are received along separate channels via cable 124 as rasterimages in memory of computer system 118 for display on screen 125 asdesired by the user. Computer system 118 via cable 124 also sendssignals to motor 174 and 175 and reads sensors associated therewith torotate filter wheels 167 and 171, respectively, in accordance with therotational position of the particular filter or opening along suchfilter wheels as desired by the user.

Different locations along tissue samples 110 or 113 are selected toprovide optical sectioned microscopic images of the sample at suchlocations presented to objective lens 128 by one or more of movingimaging head 12 as described earlier, moving stage 108 along its xand/or y axes, changing depth of the scanned beam 115 in and under thesurface of the tissue sample by moving objective lens 128 along itsoptical axis 128 a in imaging head 12, such optical axis being co-axialwith the z axis if aligned thereto as described earlier, or along z axisto change such depth by using stage 114 if present. The selection ofdifferent locations along a tissue sample 110 or 113 may be performedautomatically by computer system 118 stepwise movement along x and/or yaxes of stage 108 (and z axis of stage 114 if present), and/or stepwisemovement of objective lens 128 along its optical axis 128 a. Forexample, computer system 118 can fix the position of galvanometer mirror154 a to be stationary, and instead move stage 108 in a stepwise fashionalong the y axis to provide comparable raster scan imaging. Power andground to electronics and other components, such as laser source 146, inimaging system 12 is also provided by wires within cable 124.

Further, although imaging head 12 is described herein having an opticalsystem for capturing optically formed microscopic sectional images oftissue sample 110 or 113 operative by confocal microscopy, othermodalities for imaging optically sectioned microscopic images of samplemay be incorporated in imaging head 12 by optical coherence tomography(OCT) or interferometry, such as described in Schmitt et al., “Opticalcharacterization of disease tissues using low-coherence interferometry,”Proc. of SPIE, Volume 1889 (1993), or by a two-photon laser microscopy,such as described in U.S. Pat. No. 5,034,613.

Other positions of imaging head 12 may be provided than shown in thefigures. Also, different non-histologically tissue samples than tissuesamples 110 and 113 shown in the figures may be imaged by microscope 10.

From the foregoing description, it will be apparent that a confocalmicroscope having a positionable imaging head has been provided.Variations and modifications in the herein described microscope, andsystem and method for mounting an imaging head of such microscope inaccordance with the invention, will undoubtedly suggest themselves tothose skilled in the art. Accordingly, the foregoing description shouldbe taken as illustrative and not in a limiting sense.

The invention claimed is:
 1. A method for imaging tissue comprising:providing an imaging head having an optical system for capturingoptically formed microscopic sectional images; mounting said imaginghead to a platform using a plurality of stages; translating said imaginghead along a vertical dimension using a first of said plurality ofstages; rotating said imaging head about said vertical dimension using asecond of said plurality of stages; positioning said imaging head in afirst mode to image at least a first tissue sample disposed on a movablespecimen stage mounted upon said platform by carrying out one or more ofsaid translating and rotating steps; and positioning said imaging headin a second mode to image at least a second tissue sample disposedbeside said platform by carrying out one or more of said translating androtating steps.
 2. The method according to claim 1 further comprisingthe step of rotating said imaging head about a normal axis perpendicularto an optical axis of an objective lens of said optical system incarrying out one or more of said step of positioning said imaging headin said first mode and said step of positioning said imaging head insaid second mode.
 3. The method according to claim 2 further comprisingthe step of adjusting the tilt of said imaging head along said normalaxis in carrying out one or more of said step of positioning saidimaging head in said first mode and said step of positioning saidimaging head in said second mode.
 4. The method according to claim 1further comprising the step of moving said imaging head between beingpositioned in said first mode and being positioned in said second modeby at least carrying out said rotation step.
 5. The method according toclaim 1 wherein said mounting step further comprises mounting saidsecond of said plurality of stages to said platform, and mounting saidsecond of plurality of said stages to said first of said plurality ofstages.
 6. The method according to claim 1 further comprising the stepof focusing and collecting illumination using an objective lens of saidoptical system of said imaging head.
 7. The method according to claim 6further comprising the step of mounting said first tissue sample uponsaid specimen stage to move said first tissue sample with respect tosaid objective lens along at least x and y orthogonal axes when saidimaging head is positioned in said first mode in which said objectivelens has an optical axis that extends along a z axis perpendicular tosaid x and y axes.
 8. The method according to claim 7 further comprisingthe step of moving said objective lens along said optical axis in saidimaging head.
 9. The method according to claim 6 further comprising thestep of mounting said objective lens in said imaging head to bereplaceable with another one of said objective lens.
 10. The methodaccording to claim 6 further comprising the step of mounting said firsttissue sample upon said specimen stage to move said first tissue samplewith respect to said objective lens along at least x, y, and zorthogonal axes when said imaging head is positioned in said first modeand said objective lens has an optical axis that extends along said zaxis.
 11. The method according to claim 1 further comprising the step ofproviding a computer system connected to said imaging head to receivesignals representative of said images, wherein said computer systemshows said images on a display.
 12. The method according to claim 1wherein said step of providing said imaging head further comprisesproviding said imaging head having said optical system operative byconfocal microscopy for capturing said optically formed microscopicsectional images.